CellreportMatt - Differential Centrifugation of Tissue...

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Differential Centrifugation of Tissue Homogenates Purpose: Use of mid-speed centrifuge; homogenization; techniques utilized when using biological samples PRECAUTIONS: All buffers used are non-toxic, aqueous solutions. Always wear eye protection when operating motor-driven equipment; always ensure that tubes are of equal volume and balanced opposite of each other before starting the centrifuge. Maintain sample tubes as cold as possible throughout the lab exercise to inhibit degradation of molecules. Items Needed: Beakers for homogenate (50 ml or larger) 0.25 M sucrose 12 ml Centrifugation tubes with caps (6/group) balances and weigh boats Test tube holders (beakers) chicken liver graduated cylinders tissue homogenizer spatulas (small red ones) 50 ml tubes for homogenizing & mixing (3/group) scissors and forceps for cutting tissue pipettes/tips medicine droppers Perishable Needs: 0.25M Sucrose: MW = 342.3 Prepare 85.58 g sucrose/L Liver: Will need 6 g liver/group X 1 lb/453.6g = 0.0132 lb/group If 12 groups = 0.158 lbs total; 18 groups = 0.238 lbs total Procedure: NOTE: You will only need to retain one aliquot of each sample. KEEP SAMPLES ON ICE DURING THIS PROCEDURE AS MUCH AS POSSIBLE to reduce the action of degradative enzymes that may be released from broken lysosomes!!! At the end of this laboratory exercise, you will save 5 different biological samples. NOTE: Each step may need to be performed in duplicate depending upon the number of groups per laboratory section. If so, you will have two tubes per group until step 10. 1. Weigh 3 g of chicken liver on a balance. Place liver sample in a beaker on ice. If instructed, weigh out a second liver sample, placing material in a separate beaker. Keep all samples on ice throughout the exercise. 2. Add approximately 16 ml of 0.25 M sucrose to each tissue sample. Use scissors to cut tissue into smaller pieces. Transfer material to a 50 ml storage tube packed in a small beaker with ice. KEEP SAMPLES ON ICE THROUGHOUT THE LAB! 3. Using a tissue homogenizer, process liver tissue until no solids remain (this step may take 4-5 up/down strokes)— MAKE SURE TO WEAR EYE PROTECTION . This is the Original Homogenate. Save 5 ml of this material in a 12 mL centrifuge tube, labeled as O.H. on ice. If you prepared a second sample, combine the two homogenates together
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into one 50 mL storage tube, mix well, then save your sample. 4. Pour the remaining homogenate into two 12 ml centrifuge tubes, equally filling each tube to approximately one inch from the top of the tube. Discard any remaining homogenate. Leave the tubes uncapped and centrifuge the original homogenate sample at 2300 rpm (Rotor # JA 20.1) or 600-700 x g for 5-8-10 min. 5. Once the centrifuge has stopped, remove the supernatant from one tube into a fresh 12 mL
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CellreportMatt - Differential Centrifugation of Tissue...

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